Composition for inducing humoral anergy to an immunogen comprising a T cell epitope-deficient analog of the immunogen conjugated to a nonimmunogenic valency platform molecule

ABSTRACT

Conjugates of nonimmunogenic valency platform molecules and analogs of immunogens that possess the specific B cell binding ability of the immunogen but lack T cell epitopes and which, when introduced into individuals, induce humoral anergy to the immunogen are disclosed. Accordingly, these conjugates are useful for treating antibody-mediated pathologies that are caused by foreign or self immunogens.

RELATED APPLICATION

[0001] This application is a continuation-in-part of Ser. No.07/652,648, filed Feb. 8, 1991, the disclosures of which areincorporated herein by reference.

TECHNICAL FIELD

[0002] This invention is in the field of immunology and concernscompositions and methods for inducing humoral anergy for the purpose oftreating antibody-mediated pathologies. More specifically, the inventionrelates to conjugates of nonimmunogenic valency platform molecules andanalogs of immunogens that lack T cell epitopes.

BACKGROUND OF THE INVENTION

[0003] In order to survive in a world of pathogenic or potentiallypathogenic microorganisms, higher organisms have evolved immune systemswhich can specifically recognize virtually any foreign substance throughits characteristic molecules. This recognition frequently results in theproduction of specific proteins called antibodies which bind only to theforeign substance which induced their synthesis, causing the eliminationof the invading microorganism. Occasionally an animal's immune systemmakes antibodies which recognize some of its own molecules, generatingan autoimmune state that may affect the animal's health adversely.

[0004] The induction of specific antibodies in response to an immunogeninvolves the interaction of multiple cell types, includingthymus-derived lymphocytes (T cells), macrophages, and bonemarrow-derived lymphocytes (B cells). This is in contrast to the primary(IgM) immune response which does not include T cells. T cell dependentantigen responses are secondary responses. B cells possess surfaceimmunoglobulin by which they are able to bind immunogens, the first stepin their activation and clonal expansion. A single B cell expresses onlyone type of antigen-specific immunoglobulin. The site(s), region(s) ordomain(s) of the immunogen to which the immunoglobulin binds is called a“B cell epitope.” In the second step of B cell activation and expansion,T cells are activated through interaction with a site, region or domainof the immunogen called a “T cell epitope” which is presented by B cellsor other antigen-presenting cells. Once activated, the T cells providepositive signal(s) to the B cells to which the immunogen is bound andthey proceed to differentiate and to produce and secrete antibody.Positive signals from the T cell include the secretion of lymphokines,and/or direct contact between the B cells and T cells. T cell epitopesmay be different or more restricted in scope than B cell epitopes. Asdiscussed above, in order for an immunogen to elicit T dependentantibodies, it must have epitopes recognized by both B and T cells.

[0005] Past attempts to treat antibody-mediated pathologies haveinvolved both general and specific suppression of the immune response.General suppression has typically employed broad spectrum, nonspecificimmunosuppressants such as cyclophosphamide or steroids. Because thesenonspecific drugs suppress many aspects of the immune system, they limitits required and beneficial functions as well as the malfunction causingthe condition being treated. They are thus used only with extremecaution and subject the patient to risk from secondary infections orother undesirable side effects.

[0006] Because of the disadvantages of general immunosuppression,methods for specifically suppressing an immune response to an immunogenwithout affecting the normal functions of the immune system are highlypreferred for treating antibody-mediated pathologies. The presentinvention concerns compositions and methods for specifically suppressingthe humoral response to immunogens.

[0007] Prior attempts to induce specific immunosuppression have focusedon conjugating haptens and immunogens to nonimmunogenic polymericcarriers. Benacerraf, Katz and their colleagues used conjugates ofhaptens and antigens and copolymers of D-lysine and D-glutamic acid(formerly D-GL, hereinafter D-EK). Their initial studies involvedconjugates of the synthetic hapten 2,4-dinitrophenyl (DNP) in guineapigs and mice and showed the conjugates were capable of inducing humoralunresponsiveness. These initial studies were then extended to conjugatesof other haptens and conjugates of immunogens. While the results withhaptens were repeatable, and although their patents (U.S. Pat. Nos.4,191,668 and 4,220,565) allege the approach is effective in inducingtolerance to immunogens, subsequent work has shown that conjugates ofD-EK and immunogens do not provide a means for inducing humoralunresponsiveness to the immunogen. For instance, Liu et al., J. Immun.(1979) 123:2456-2464, report that subsequent studies of those conjugatesdemonstrate that the conjugates “do not induce unresponsiveness at thelevel of protein specific B cells.” Similarly, Butterfield et al., J.Allergy Clin. Immun. (1981) 67:272-278, reported that conjugates ofragweed immunogen and D-EK actually stimulated both IgE and IgGresponses to the immunogen.

[0008] This subsequent work and other data dealing with conjugates ofnonimmunogenic polymers and immunogens (Saski et al., Scand. J. Immun.(1982) 16:191-200; Sehon, Prog. Allergy (1982) 32:161-202; Wilkinson etal., J. Immunol. (987) 139:326-331, and Borel et al., J. Immunol.Methods (1990) 126:159-168) appear to indicate that the anergy, if any,obtained with such conjugates is due to suppression by T cells todirectly suppress the immune response.

[0009] Several other references deal with conjugates of nonimmunogenicpolymers and DNA. See U.S. Pat. No. 4,191,668; U.S. Pat. No. 4,650,625;J. Clin. Invest. (1988) 82:1901-1907; and commonly owned U.S. patentapplication Ser. No. 07/494,118. As a whole, these references indicatethat these DNA conjugates may suppress the production of antibodies tothis lupus autoimmunogen. It should be noted in this regard that DNA isnot immunogenic and does not possess T cell epitopes.

[0010] In sum, applicants believe the prior art shows that antibodyproduction to conjugates of nonimmunogenic stable polymers and haptensor DNA, neither of which have T cell epitopes, may provide B cellunresponsiveness. Applicants also believe that conjugates of immunogensdo not provide B cell unresponsiveness but may activate T cells todirectly suppress the immune response.

DISCLOSURE OF THE INVENTION

[0011] The present invention resides in the discovery that the failureof the prior conjugates of nonimmunogenic polymers and immunogens toinduce B cell anergy (unresponsiveness) was due to the fact that theimmunogens contained both B and T cell epitopes and that if the latterwere eliminated, the conjugate would be effective for inducing B cellanergy.

[0012] Accordingly, one aspect of the invention is a composition forinducing specific B cell anergy to an immunogen comprising a conjugateof a nonimmunogenic valency platform molecule and an analog of theimmunogen that (a) binds specifically to B cells to which the immunogenbinds and (b) lacks the T cell epitope(s) of the immunogen.

[0013] Pharmaceutical compositions of the above-described conjugates andpharmaceutically acceptable carriers or vehicles are another aspect ofthe invention.

[0014] A further aspect of the invention is a method of inducingspecific B cell anergy to an immunogen in an individual comprisingadministering to the individual an effective amount of theabove-described conjugate.

[0015] Yet another aspect of the invention is a method of treating anindividual for an antibody-mediated pathology in which undesiredantibodies are produced in response to an immunogen comprisingadministering to the individual a therapeutically effective amount ofthe above-described conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 graphically illustrates the detection of B cell epitopes inimmunized CAF1 mice as described in Example 1. The unconjugated 17 merpeptide (L-42) and L-42 and L-53 D-EK conjugates were tested for B cellepitopes with anti-MIR sera. Unconjugated L-42 and L-53 were tested withnormal mouse serum (NMS) as a control.

[0017]FIG. 2, similarly, illustrates the detection of T cell epitopes asdescribed in Example 1.

[0018]FIG. 3 illustrates the suppression of antibodies to peptide “L-53”as described in Example 1.

[0019]FIGS. 4 and 5 are graphs of the results described in Example 4.

[0020]FIG. 6 compares the level of T cell proliferation induced bymelittin peptides.

[0021]FIG. 7 compares the levels of anti-melittin peptide 2 antibodiesproduced in mice treated with Conjugate 2 versus the control micetreated with formulation buffer.

[0022]FIG. 8 compares the levels of anti-melittin antibodies produced inmice treated with Conjugate 2 versus the control mice treated withformulation buffer.

[0023]FIG. 9 compares the levels of anti-melittin peptide 2antibody-forming cells in mice treated with Conjugate 2 versus thecontrol mice treated with formulation buffer.

[0024]FIG. 10 illustrates that Conjugate 4, a conjugate of peptide #5which contains a T cell epitope, was not a tolerogen.

[0025]FIG. 11 illustrates melittin conjugates within the presentinvention.

MODES FOR CARRYING OUT THE INVENTION

[0026] As used herein the term “B cell anergy” intends unresponsivenessof those B cells requiring T cell help to produce and secrete antibodyand includes, without limitation, clonal deletion of immature and/ormature B cells and/or the inability of B cells to produce antibody.“Unresponsiveness” means a therapeutically effective reduction in thehumoral response to an immunogen. Quantitatively the reduction (asmeasured by reduction in antibody production) is at least 50%,preferably at least 75%, and most preferably 100%.

[0027] “Antibody” means those antibodies which are T cell dependent.

[0028] As used herein the term “immunogen” means a chemical entity thatelicits a humoral immune response when injected into an animal.Immunogens have both B cell epitopes and T cell epitopes.

[0029] The term “analog” of an immunogen intends a molecule that (a)binds specifically to an antibody to which the immunogen bindsspecifically and (b) lacks T cell epitopes. Although the analog willnormally be a fragment or derivative of the immunogen and thus be of thesame chemical class as the immunogen (e.g., the immunogen is apolypeptide and the analog is a polypeptide), chemical similarity is notessential. Accordingly, the analog may be of a different chemical classthan the immunogen (e.g., the immunogen is a carbohydrate and the analogis a polypeptide) as long as it has the functional characteristics (a)and (b) above. The analog may be a peptide, carbohydrate, lipid,lipopolysaccharide, nucleic acid or other biochemical entity. Further,the chemical structure of neither the immunogen nor the analog need bedefined for the purposes of this invention.

[0030] An analog of an immunogen may also comprise a “mimotope.” Theterm “mimotope” intends a synthetic molecule which competitivelyinhibits the antibody from binding the immunogen. Because itspecifically binds the antibody, the mimotope is considered to mimic theantigenic determinants of the immunogen. Like an analog of an immunogen,a mimotope (a) binds specifically to an antibody to which the immunogenbinds specifically and (b) lacks T cell epitopes.

[0031] An analog of an immunogen may also comprise an “aptamer.” Theterm “aptamer” intends a synthetic oligonucleotide which competitivelyinhibits the antibody from binding the immunogen. Like an analog of animmunogen, an aptamer (a) binds specifically to an antibody to which theimmunogen binds specifically and (b) lacks T cell epitopes.

[0032] As used herein “valency platform molecule” means a nonimmunogenicmolecule containing sites which facilitate the attachment of a discreetnumber of analogs of immunogens.

[0033] “Nonimmunogenic” is used to describe the valency platformmolecule and means that the valency platform molecule elicitssubstantially no immune response when it is administered by itself to anindividual.

[0034] As used herein “individual” denotes a member of the mammalianspecies and includes humans, primates, mice and domestic animals such ascattle and sheep, sports animals such as horses, and pets such as dogsand cats.

[0035] Immunogens that are involved in antibody-mediated pathologies maybe external (foreign to the individual) immunogens such as allergens, Rhhemolytic disease (D immunogen), biological drugs, including nativebiological substances foreign to the individual such as therapeuticproteins, peptides and antibodies, and the like or self-immunogens(autoimmunogens) such as those associated with thyroiditis(thyroglobulin), stroke (cardiolipin), male infertility (α-sperm),myasthenia gravis (acetylcholine receptor) and rheumatic fever(carbohydrate complex).

[0036] Analogs to such immunogens may be identified by screeningcandidate molecules to determine whether they (a) bind specifically toserum antibodies to the immunogen and (b) lack T cell epitopes. Specificbinding to serum antibodies may be determined using conventionalimmunoassays and the presence or absence of T cell epitopes may bedetermined by conventional T cell activation assays. In this regard ananalog which “binds specifically” to serum antibodies to the immunogenexhibits a reasonable affinity thereto. The presence or absence of Tcell epitopes may be determined using the tritiated thymidineincorporation assay described in the examples. The presence of T celleptiopes can also be determined by measuring secretion of T cell-derivedlymphokines by methods well known in the art. Analogs that fail toinduce statistically significant incorporation of thymidine abovebackground are deemed to lack T cell epitopes. It will be appreciatedthat the quantitative amount of thymidine incorporation may vary withthe immunogen. Typically a stimulation index below about 2-3, moreusually about 1-2, is indicative of a lack of T cell epitopes.

[0037] A normal first step in identifying useful analogs is to prepare apanel or library of candidates to screen. For instance, in the case ofprotein or peptide analogs, libraries may be made by synthetic orrecombinant techniques such as those described by Geysen et al. inSynthetic Peptides as Antigens; Ciba Symposium (1986) 119:131-149;Devlin et al., Science (1990) 249:404-406; Scott et al., Science (1990)249:386-390; and Cwirla et al., PNAS USA (1990) 87:6378-6382. In onesynthetic technique, peptides of about 5 to 30 amino acids aresynthesized in such a manner that each peptide overlaps the next and alllinear epitopes are represented. This is accomplished by overlappingboth the carboxyl and amino termini by one less residue than thatexpected for a B cell epitope. For example, if the assumed minimumrequirement for a B cell epitope is six amino acids, then each peptidemust overlap the neighboring peptides by five amino acids. In thisembodiment, each peptide is then screened against antisera producedagainst the native immunogen, either by immunization of animals or frompatients, to identify the presence of B cell epitopes. Those moleculeswith antibody binding activity are then screened for the presence of Tcell epitopes as described in the examples. The molecules lacking T cellepitopes are useful as analogs in the invention.

[0038] If the T cell epitope(s) of an immunogen are known or can beidentified, random T cell screening of candidate analogs is notnecessary. In such instances, the T cell epitope(s) may be altered(e.g., by chemical derivatization, or elimination of one or morecomponents of the epitope) to render them inoperative or be eliminatedcompletely, such as, for instance, in the case of peptides, by syntheticor recombinant procedures.

[0039] Mimotopes and aptamers are synthesized by conventional methodsand are screened in the same manner as other analogs of immunogens.

[0040] The analogs are coupled to a nonimmunogenic valency platformmolecule to prepare the conjugates of the invention. Preferred valencyplatform molecules are biologically stabilized, i.e., they exhibit an invivo excretion half-life often of hours to days to months to confertherapeutic efficacy, and are preferably composed of a synthetic singlechain of defined composition. They will normally have a molecular weightin the range of about 200 to about 200,000, usually about 200 to about20,000. Examples of valency platform molecules within the presentinvention are polymers such as PEG, poly-D-lysine, polyvinyl alcohol,polyvinylpyrollidone, immunoglobulins, and D-EK. Preferred polymers arebased on polyethylene glycols (PEGS) having a molecular weight of about200 to about 8,000. Other preferred polymers are D-EKs having amolecular weight of about 5,000 to about 30,000, and an E:K (D-glutamicacid:D-lysine) mole ratio of approximately 60:40, as described in U.S.patent application Ser. No. 07/494,118, referenced above.

[0041] Conjugation of the analog to the valency platform molecule may beeffected in any number of ways, typically involving one or morecrosslinking agents and functional groups on the analog and valencyplatform molecule.

[0042] Polypeptide analogs will contain amino acid sidechain groups suchas amino, carbonyl, or sulfhydryl groups that will serve as sites forcoupling the analog to the carrier. Residues that have such functionalgroups may be added to the analog if the analog does not already containsame. Such residues may be incorporated by solid phase synthesistechniques or recombinant techniques, both of which are well known inthe peptide synthesis arts. In the case of carbohydrate or lipidanalogs, functional amino and sulfhydryl groups may be incorporatedtherein by conventional chemistry. For instance, primary amino groupsmay be incorporated by reaction with ethylenediamine in the presence ofsodium cyanoborohydride and sulfhydryls may be introduced by reaction ofcysteamine dihydrochloride followed by reduction with a standarddisulfide reducing agent. In a similar fashion the valency platformmolecule may also be derivatized to contain functional groups if it doesnot already possess appropriate functional groups. With specificreference to conjugating peptide analogs and D-EK or other proteinaceousvalency platform molecules, coupling is preferably carried out using aheterobifunctional crosslinker, such assulfosuccinimidyl(4-iodoacetyl)aminobenzoate, which links the ε aminogroup on the D-lysine residues of D-EK to a sulfhydryl side chain froman amino terminal cysteine residue on the peptide to be coupled. Thismethod is usually carried out such that an average of 3 to 5 analogmolecules are coupled to each D-EK molecule and the average molecularweight of the D-EK prior to coupling is 5,000 to 30,000 daltons.

[0043] The conjugates will normally be formulated for administration byinjection (e.g., intraperitoneally, intramuscularly, etc.). Accordingly,they will typically be combined with pharmaceutically acceptablevehicles such as saline, Ringer's solution, dextrose solution, and thelike. The conjugate will normally constitute about 0.01% to 10% byweight of the formulation. The conjugate is administered to anindividual in a “therapeutically effective amount”, i.e., an amountsufficient to produce B cell anergy to the involved immunogen and effectprophylaxis, improvement or elimination of the antibody-mediatedcondition being addressed. The particular dosage regimen, i.e., dose,timing and repetition, will depend on the particular individual and thatindividual's medical history. Normally, a dose of about 10 μg to 1 mgconjugate/kg body weight will be given, daily for three consecutivedays. Other appropriate dosing schedules would be 3 doses per week, orone dose per week, or one dose every two to four weeks, or one dose on amonthly or less frequent schedule depending on the individual or thedisease state. Repetitive administrations, normally timed according to Bcell turnover rates, may be required to achieve and/or maintain a stateof humoral anergy. Such repetitive administrations will typicallyinvolve treatments of up to 1 mg/kg of body weight every 30 to 60 days,or sooner, if an increase in antibody titer is detected. Alternatively,sustained continuous release formulations of the conjugates may beindicated for some pathologies. Various formulations and devices forachieving sustained release are known in the art.

[0044] Anti-T helper cell treatments may be administered together withthe conjugates. Such treatments usually employ agents that suppress Tcells such as steroids or cyclosporin.

[0045] The following examples are intended to further illustrate theinvention and its uniqueness. These examples are not intended to limitthe scope of the invention in any manner.

EXAMPLE 1 B Cell Anerqy to the Acetylcholine Receptor

[0046] Preparation of Peptides and D-EK/Peptide Conjugates:

[0047] The α-subunit of the acetylcholine receptor of Torpedocalifornicus is described by Stroud, R. M., and Finer-Moore, J., Ann.Rev. Cell Biol. (1985) 1:317:351, and Sumikawa, K., et al., Nucl. AcidsRes. (1982) 10:5809-22. The peptide defined by residues 47-127 of thatα-subunit is called the major immunogenic region (MIR).

[0048] Two peptides, L-42 and L-53, corresponding to residues 61-77 and112-127 of that α-subunit, were synthesized using conventionalsolid-phase methods and purified to homogeneity by HPLC. An aminoterminal cysteine was added to each sequence for the purpose ofattachment of the peptide to D-EK via a thio ether linkage.

[0049] Each peptide (40 mg) was dissolved in 0.1 M sodium borate buffer,pH 9.0. The solution was reacted with citraconic anhydride (400 μL) atroom temperature; the pH was maintained above 7.0 by addition of 1 MNaOH. The solution was then made 20 mM in dithiothreitol and was warmedat 37° C. for 20 minutes to reduce the peptide. The mixture was quicklydesalted over G-10 Sephadex columns which were equilibrated with 0.1Msodium borate, pH 7.0.

[0050] D-EK (200 mg, weight average molecular weight≅10,000-30,000) wasdissolved in 2.0 mL of 0.1M sodium borate. Sulfosuccinimidyl(4-iodoacetyl) aminobenzoate (SSIAB, 10 mg, Pierce Chemical) was addedto the mixture and the mixture was reacted for 90 minutes at roomtemperature in the dark. The mixture was then desalted over a 10 mL G-25column, equilibrated with 0.1M sodium borate, pH 7.0.

[0051] The desalted SSIAB-D-EK was mixed with the reduced and desaltedpeptide and reacted overnight. The resulting conjugate was placed indialysis tubing with a 14 Kd cutoff and was dialyzed against 5% aceticacid to remove citraconyl groups. The dialysis buffer was changed tophosphate-buffered saline and the dialysis continued.

[0052] Detection of B Cell Epitopes:

[0053] CAF1 mice were obtained and housed at the La Jolla Pharmaceuticalanimal facility according to National Institutes of Health guidelines.CAF1 mice were immunized (day 0) intraperitoneally (i.p.) with 50 μg ofrecombinant torpedo MIR absorbed onto alum plus B. pertussis vaccine (B.pertussis vaccine obtained from Michigan Department of Public Health,Lansing, Mich.) (Iverson, G. M., (1986) Handbook of ExperimentalImmunology, Vol. 2, p. 67, D. M. Weir ed., Blackwell ScientificPublications, Palo Alto, Calif.). The mice received a booster injectionof the same protein in saline, i.p., on day 21 and were bled from thetail vein on day 28. Sera from these mice (anti-MIR sera) were used toscreen peptides L-42 and L-53 for the presence of B cell epitopes, asfollows. The sera were added to the wells of microtitration plates whichwere coated with 10 μg/mL of the indicated peptide conjugates. Theplates were incubated at 37° C. for one hour, washed 3 times, 100 μl ofalkaline phosphatase-conjugated goat anti-mouse antibody was added,incubated at 37° C. for one hour, washed 3 times, and 100 μl ofdeveloper (substrate) was added to each well. The plates were incubatedat room temperature for 30 minutes and the amount of color in each wellwas determined in a Titertek® Multiskan microplate reader. Results areillustrated graphically in FIG. 1. The curve labelled “L42 or L53, NMS”contains the values obtained using normal mouse serum (NMS) instead ofthe anti-MIR sera on plates coated with either L42 or L53. As shown inFIG. 1, both peptides reacted specifically with antibodies from theimmunized mice indicating the presence of B cell epitopes on bothpeptides.

[0054] Detection of T Cell Epitopes:

[0055] T cell activation was assayed by the general procedure ofBradley, M. L., (1980) in Mishell and Shigii, eds., Selected Methods inCellular Immunoloqy (W. H. Freeman and Co., San Francisco, Calif.), p.164. CAF1 mice were obtained and housed at the La Jolla Pharmaceuticalanimal facility according to National Institutes of Health guidelines.CAF1 mice were immunized in the footpad with 50 μg MIR in CompleteFreund's Adjuvant (CFA) on day 0. On day 7 the popliteal lymph nodeswere removed and placed in culture in microtiter plates using 5×10⁵cells per well. The peptides or peptide-DEK conjugate were added to thecultures, and on day 4, 1 μCi of tritiated thymidine was added to eachwell to measure proliferation of T cells. The cultures were harvested onday 5 with a Skatron® cell harvester. The amount of incorporated³H-thymidine was determined in a Beckman L6800® liquid scintillationcounter. The stimulation index was calculated by dividing the CPMincorporated with peptide by the CPM incorporated from cultures withoutany peptide. A stimulation index>2-3 was indicative of the presence of aT cell epitope on the peptide added to the well. As shown in FIG. 2,L-42 but not L-53 possessed T cell epitopes in this assay.

[0056] Induction of B Cell Anergy to L-53 by L-53-D-EK Conjugate:

[0057] CAF1 mice were obtained and housed at the La Jolla Pharmaceuticalanimal facility according to National Institutes of Health guidelines.CAF1 mice were immunized with 50 μg of MIR, i.p., absorbed onto alumplus B. pertussis vaccine on day 0. On days 21, 22 and 23 the mice (6mice per group) received 10 or 100 μg of either L-42-D-EK conjugate orL-53-D-EK conjugate. One group received only saline. On day 28 all micereceived a booster injection of MIR in saline and on day 35 all micewere bled and assayed for the presence of antibodies to L-42 and L-53 intheir sera, using an ELISA assay as described above with respect toFIG. 1. The results for antibodies to L42 are shown in FIG. 3A and forantibodies to L53 are shown in FIG. 3B. The L-53 conjugate, which didnot contain a T cell epitope, suppressed antibody formation to L-53 butnot to L-42. The L-42 conjugate, which contained a T cell epitope, didnot suppress the antibody response to either L-42 or L-53, but rathermay have increased antibody production to L-42. The antibody titers areexpressed as a percent of a standard sera. The P values were determinedby a Student t test comparing each dose to the saline control.

EXAMPLE 2 Failure of Ovalbumin-D-EK Conjugate to Induce B Cell Anergy toOvalbumin

[0058] This example is further evidence that conjugates of immunogensand D-EK do not induce B cell anergy.

[0059] Synthesis of Ovalbumin-D-EK Conjugate:

[0060] Chicken egg ovalbumin (ova; 50 mg) was dissolved in 5 mL of 0.1Msodium borate buffer, pH 9.0, containing 10 mM EDTA. After the additionof 3.0 mg of 2-iminothiolane (Traut's reagent), the mixture was reactedfor 2.5 hours at room temperature. D-EK (54 mg), dissolved in 0.5 Msodium borate, pH 9.0, at a concentration of 100 mg/mL, was reacted withSSIAB (18 mg; Pierce Chemical) for 2.5 hours in the dark, at roomtemperature. The two reaction mixtures described above were desaltedseparately on G-25 columns (Pharmacia; 10 mL column volume, equilibratedwith 0.1 M sodium borate, pH 9.0) and the excluded fractions werecombined and reacted for 16 hours at 40° C., in the dark. The reactionproduct was fractionated by gel filtration over Sephacryl S-200 (490 mL,Pharmacia) columns, equilibrated with 0.2 M ammonium bicarbonate.Fractions containing conjugate, as assessed by polyacrylamide gelelectrophoresis, in the presence of sodium dodecyl sulfate (SDS-PAGE),were pooled and dried under vacuum. The dried material was reacted with0.8 mL of citraconic anhydride, maintaining the pH between 7 and 9 bythe addition of 1M NaOH, in order to efficiently separate conjugatedovalbumin from unreacted protein. The citraconylated conjugate wasrechromatographed over S-200, and fractions containing high molecularweight material (>80,000 daltons), as assessed SDS-PAGE, were used forbiological studies.

[0061] Chicken Ovalbumin, when Conjugated to D-EK, does not Induce BCell Anergy in Mice Immunized to Chicken Ovalbumin:

[0062] CAF1 mice were obtained and housed at the La Jolla Pharmaceuticalanimal facility according to National Institutes of Health guidelines.Female CAF₁ mice were primed with ova (100 μg/mouse, i.p.) precipitatedon alum, with B. pertussis vaccine added as an adjuvant. Sixteen weekslater, the mice were divided into two groups of six mice each. One group(control) was treated with saline, and the second group was injectedwith a conjugate of ova and D-EK (ova-D-EK; 200 μg/mouse/day, i.p.). Themice were dosed on three successive days. One week after the first dose,the mice in both groups were boosted, i.p., with ova in saline (100μg/mouse). One week later, the mice were bled from a tail vein. Theplasma was harvested and assayed for the amount of anti-ova antibodiesby an ELISA assay. As shown in Table 1, the ova-D-EK conjugate did notsuppress the anti-ova response. TABLE 1 percent of Anti-Ova Standard¹Group Treatment Serum ± S.D. 1 saline 70.7 ± 36   2 ova-D-EK 160.2 ±167  

EXAMPLE 3 Failure of MIR-D-EK Conjugate to Induce B Cell Anergy to MIR

[0063] This example is still further evidence that conjugates ofimmunogens and D-EK do not induce B cell anergy.

[0064] Synthesis of MIR-D-EK Conjugate:

[0065] MIR was modified on its carboxyl-terminus to include a sequenceof 8-amino acids (Arg-Ser-Lys-Ser-Lys-Ser-Lys-Cys (SEQ. ID NO.: 1)). Theamino-terminus was extended by one amino acid, proline. Purifiedmodified MIR (250 mg) was reduced with 100 mM dithiothreitol and wasdesalted over Sephadex G-25 (Pharmacia), equilibrated with 0.1 M sodiumborate buffer, pH 9.0, containing 10 mM EDTA. D-EK (400 mg) was reactedwith SSIAB (29 mg) as in the previous examples. The product was desaltedover G-25. The excluded volumes from the modified MIR and D-EK G-25column runs were combined and reacted at 4° C. for 16 hours, in thedark. Excess SSIAB groups were quenched with 2-mercaptoethanol, and thereaction mixture was concentrated to 20 mL over a PM-10 membrane (AmiconCorporation). The mixture was treated with 1.0 mL of citraconicanhydride and chromatographed over S-300 (Pharmacia; 1.8 L),equilibrated with 5% ammonium hydroxide. Fractions containing two ormore modified MIR groups per D-EK, as assessed by SDS-PAGE, were pooledand used for biological studies.

[0066] MIR-D-EK Conjugate Contains T cell Epitopes Recognized by RatsImmunized with MIR from the Same Species:

[0067] T cell activation was assayed by the general procedure ofBradley, supra. Female Lewis rats were immunized in the footpad with MIR(50 μg) in complete Freund's adjuvant (CFA) on day 0. On day 7, thepopliteal lymph nodes were removed and placed in culture in microtiterplates using 5·10⁵ cells per well. MIR-D-EK was added, and, after fourdays of culture, the wells were pulsed with tritiated thymidine (1-μCi)to measure proliferation of T cells. The cultures were collected after 5days of culture with a Skatron™ cell harvester. The amount ofincorporated ³H-thymidine was determined by scintillation spectrometry.The stimulation index was calculated by dividing the counts incorporatedin the absence of the conjugate. A stimulation index of greater than 2-3was considered indicative of the presence of a T cell epitope on theadded conjugate. The stimulation index was 4 or greater at allconcentrations of MIR-D-EK tested (10 μg/mL to 400 mg/mL), proving thatT cells from MIR-immunized rats recognize T cell epitopes on theMIR-D-EK conjugate in this assay.

[0068] MIR-D-EK does not Induce B Cell Anergy in Rats Immunized withMIR:

[0069] Female Lewis rats were primed with MIR (100 μg/rat) in CFA. Sixmonths later, the rats were divided into three groups of three ratseach. One group was treated with saline (control) and the other twogroups were treated with MIR-D-EK (100 μg/rat, i.p.) on three successivedays. After one week, the rats in the control group and one group thathad been treated with MIR-D-EK were boosted with recombinant MIR (1000μg/rat, i.p.) in saline. One week later, all three groups of rats werebled from the tail vein. The plasma was harvested and assayed for theamount of anti-MIR antibodies by an ELISA assay. Table 2 below reportsthe data from those assays. TABLE 2 μL equivalence (% of standardanti-MIR¹) P. vs. Group Treatment MIR Boost (mean ± S.D.) Group 1 1Saline Yes 130.5 ± 74.7  2 MIR-D-EK Yes 85.5 ± 31.1 0.195 3 MIR-D-EK No230.6 ± 31   0.049

[0070] As shown in Table 2, the data on Group 1 animals (saline control)indicate that MIR itself is an immunogen. The data for the Group 2 and 3animals indicate that the MIR-D-EK conjugate did not suppress theanti-MIR response. In fact, MIR-D-EK boosted the anti-MIR response inGroup 3.

[0071] These tests, taken together with the results of Example 1 showthat the moiety conjugated to D-EK will cause anergy in B cellsrecognizing that moiety if the moiety either does not contain a T cellepitope or is not recognized by T cells.

EXAMPLE 4 Tests with Conjugate of L-42 and KLH

[0072] Synthesis of L42 Peptide-KLH Conjugate:

[0073] Reduced L-42 (see Example 1) was conjugated to keyhole limpethemocyanin (KLH) using thioether chemistry similar to that describedabove with respect to D-EK.

[0074] L-42 does not Activate T Cells in Mice Immunized with L-42-KLH:

[0075] Activation of T cells by peptides was measured by the generalprocedure of Bradley, supra. Female CAF₁ mice were immunized in thefootpad with L-42 peptide conjugated KLH (L-42-KLH; 50 μg) in CFA on day0. On day 7, the popliteal lymph nodes were removed and placed inculture in microtiter plates, at a cell density of 5·10⁵ cells/well.Peptides were added, and, after four days of culture, the wells werepulsed with 1 μCi of tritiated thymidine to measure proliferation of Tcells. The cultures were collected after 5 days of culture with aSkatron™ cell harvester. The amount of incorporated ³H-thymidine wasdetermined by scintillation spectrometry. The stimulation index wascalculated by dividing the counts incorporated in the absence ofpeptide. An index of greater than 2-3 is indicative of the presence of aT cell epitope on the added peptide.

[0076] The data in FIG. 4 demonstrate that the L-42 did not stimulatethe growth of T cells taken from L-42-KLH-immunized mice, and thereforedid not contain an epitope(s) recognized by T cells induced byimmunization with L-42-KLH.

[0077] L-42-D-EK conjugate induces a B cell anergy in mice immunized toL-42-KLH:

[0078] CAF₁ mice were primed with 100 μg/mouse of L-42-KLH on alum plusB. pertussis vaccine as an adjuvant. Three weeks later, the mice weredivided into groups of six mice each. One group was treated by i.p.injections on three successive days with saline (control); the othergroups were similarly treated with various doses of L-42-D-EK (i.p.).Five days later, all mice were boosted with L-42-KLH (50 μg/mouse), and,after a wait of one week, they were bled from the tail vein. The plasmawas harvested and assayed for the amount of anti-L-42 and anti-KLHantibodies by ELISA assays. Data are expressed as a percent of astandard serum. An asterisk indicates that a data point wassignificantly different from the control as determined by a Student ttest.

[0079] The data in FIG. 5 demonstrate that the anti-L-42 response, butnot the anti-KLH response, was suppressed in this assay by the L-42-D-EKconjugate. Thus, the studies summarized in Example 1 and these datademonstrate the L-42-D-EK induces B cell anergy when the mice areimmunized in a manner that does not induce the proliferation of T cellclones that recognize the L-42 peptide. This is in contrast to Example 1where L-42-D-EK did not induce B cell anergy in animals that wereimmunized with an immunogen (MIR) which induced T cells that recognizedthe L-42 peptide.

EXAMPLE 5 Preparation of Melittin Peptides and Conjugates

[0080] The melittin molecule, composed of 26 amino acids, is one of themajor components of bee venom. One third of the bee venom sensitiveindividuals have melittin specific antibodies. Melittin is highlyimmunogenic in some mouse strains (Balb/c, CAF1). The majority (>80%) ofmelittin-specific antibodies in the responder mouse strains bind a Bcell epitope which is the C-terminal heptapeptide of melittin.

[0081] Melittin

[0082]H₂N-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH₂

[0083] Melittin Peptides for T cell Stimulation

[0084] Melittin Peptide #1.

[0085] Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly (“7 mer”) (SEQ. ID NO.: 2).

[0086] Melittin Peptide #2.

[0087] Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly (“8 mer”) (SEQ. ID NO.: 3).

[0088] Melittin Peptide #3.

[0089] Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly (“9 mer”) (SEQ ID NO.:4).

[0090] Melittin Peptide #4.

[0091] Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly (“10 mer”) (SEQ. IDNO.: 5).

[0092] Melittin Peptide #5.

[0093] Cys-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly (“10 mer+C”)(SEQ. ID NO.: 6).

[0094] Peptide Synthesis

[0095] Melittin peptides were synthesized using standard Fmoc chemistrytechniques on a glycine resin (Advanced ChemTech #SG5130 or equivalent(Advanced ChemTech, 2500 Seventh Street Road, Louisville, Ky.) using 2.3M excess amino acid derivatives for each coupling step. Completion ofthe coupling was monitored with bromphenol blue and confirmed withninhydrin.

[0096] Melittin Peptides Used in Conjugations

[0097] Melittin Peptide #5.

[0098] H₂N-Cys-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly-CO₂H (SEQ. IDNO.: 7).

[0099] Melittin Peptide #6—(Peptide #2+C).

[0100] H₂N-Cys-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly-CO₂H (SEQ. ID NO.:8).

[0101] Melittin Peptide #7.

[0102] H₂N-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Lys-Cys-Gly-CO₂H (SEQ. IDNO.: 9).

[0103] Melittin Peptide #8.

[0104] (H₂N-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln)₂-Lys-Cys-Gly-CO₂H (SEQ. IDNO.: 10).

[0105] A cysteine was added as required for coupling certain peptidesvia a thioether bond to the valency platform molecule. Peptides werepurified by reversed phase HPLC following synthesis and lyophilized todryness. The appropriate amount of peptide was then weighed out for eachconjugation.

[0106] Reduction of Preformed Disulfide Bonds:

[0107] Tributylphosphine Method

[0108] All buffers were sparged with helium. The peptide was dissolvedin a minimal volume (approximately 10 to 20 mg/mL) of 0.05 M NaHCO₃ (pH8.25). A 1 mL solution of 0.7 M tributylphosphine (TBP; MW=202.32g/mole; d-0.812 g/mL) was prepared by adding 174 μL of TBP to 826 μL ofisopropanol (iPrOH). Then, 1:1 equivalents of TBP were added to thepeptide solution prepared as described above, mixed well, and allowed toreact for 30 minutes to 1 hour with occasional mixing to keep TBPdissolved and/or dispersed in the solution. Complete reduction wasconfirmed by HPLC.

[0109] Preparation of Valency Platform Molecules #3 or #5:

[0110] Compound 1-[3,5-Bis-(iodoacetamido)benzoic acid]:

[0111] 2.93 g (8.28 mmol, 2.2 eq) of iodoacetic anhydride was added to astirred suspension of 572 mg (3.76 mmol) of 3,5-diaminobenzoic acid in19 mL of dioxane at room temperature under N₂ atmosphere. The mixturewas stirred, covered with foil for 20 hours and partitioned between 50mL of EtOAc and 50 mL of 1 N HCl solution. The EtOAc layer was washedwith brine, dried over MgSO₄, filtered, and concentrated on a rotaryevaporator to give 3.3 g of tan solid. The material was purified bysilica gel chromatography (94/5/1 CH₂Cl₂/MeOH/HOAc) to yield 992 mg(54%) of compound 1 as a white solid: NMR (DMSO) 3.84 (s, 4H), 7.91 (s,2H), 8.14 (s, 1H), 10.56 (s, 2H).

[0112] Compound 2-[3,5-Bis-(iodoacetamido)benzoyl chloride]:

[0113] 117 μL (1.6 mmol, 190 mg) of SOCl₂ was added to a solution of 390mg (0.8 mmol) of 1 in 34 mL of THF. The mixture was refluxed under N₂atmosphere until all solids had dissolved (approximately 30 minutes) togive a clear red-brown solution. The mixture was concentrated on therotary evaporator and placed under vacuum to provide crude compound 2 asa foamy solid which was used directly in the next step.

[0114] Compound 3-[N,N′-Bis-(3,5-bis-(iodoacetamido)benzoyl) derivativeof α,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol]:

[0115] 570 mg of α,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol(0.16 mmol, 3350 g/mol, Sigma) was placed in a tared flask. Toluene (20mL) was added and water was removed by azeotropic distillation. Theresidue was dried under vacuum to give 549 mg of solid and dissolved in4 mL THF with 89 μL (0.64 mmol) of diisopropylethylamine. The crude acidchloride was dissolved in 4 mL anhydrous THF and added to the mixtureover 30 seconds under N₂. The mixture was stirred for 16 hours at roomtemperature and partitioned between 25 mL of 0.1 N HCl and 25 mL ofCH₂Cl₂. The aqueous layer was again extracted with CH₂Cl₂ and theorganic layers were combined, washed with 25 mL of H₂O, followed by 50mL of at NaHCO₃ solution. The organic layers were dried with Na₂SO₄,filtered, and concentrated to give 784 mg of orange oil. Silica gelchromatography (9/1 CH₂Cl₂/MeOH) yielded 190 mg of colorless oil whichwas crystallized from hot EtOH/Et₂O, collected on sintered glass filterunder N₂ pressure, and dried under vacuum to provide 177 mg of compound3 as a white solid: NMR (CDCl₃ 3.40 (bd m, 8H), 3.59 (bd s,(CH₂CH₂O)_(n), integral too large to integrate in relation to otherintegrals), 3.80 (bd m, 4H), 3.91 (s, 8H), 7.49 (brd m, 2H), 7.77 (bd m,2H), 7.82 (bd s, 4H), 8.27 (bd s, 2H), 8.90 (bd m, 4H): iodoacetyldetermination (European Journal of Biochemistry (1984) 140:63-71):Calculated, 0.92 mmol/g; Found, 0.96 mmol/g.

[0116] Compound 4-[4(iodoacetamido)benzoic acid:

[0117] This compound was prepared as described by Weltman, J. K., 1983Biotechniques 1:148-152. Briefly, 708 mg (2.0 mmol) of iodoaceticanhydride was added to a solution of 137 mg (1.0 mmol) ofpara-aminobenzoic acid in 10 mL of dioxane. The mixture was stirred inthe dark for 18 hours and partitioned between 25 mL of H₂O and 25 mL ofEtOAc. The EtOAc layer was washed with saturated NaCl solution, dried(MgSO₄), filtered and concentrated to yield 797 mg of a peach coloredsolid. Recrystallization from hexanes/EtOAc yielded 221 mg (72%) of4-(iodoacetamido)benzoic acid as a white solid: mp 220-230°; ¹H NMR(DMSO) d 3.86 (s, 2H), 7.68 (d, 2H), 7.91 (d, 2H), 10.60 (s, 1H).

[0118] Compound 5-[4-(iodoacetamido)benzoyl derivative ofα,ω-bis-(N-2-aminoethylcarbamolyl)polyethyleneglycol:

[0119] 188 mg (0.909 mmol) of dicyclohexylcarbodiimide was added to asolution of 185 mg (0.606 mmol) of 4-(iodoacetamido)benzoic acid and 406mg (0.121 mmol) of α,ω-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol(Sigma Chemical Co., St. Louis, Mo., dried by azeotropic distillationwith toluene) in 2 mL of THF. The mixture was stirred for 2 hours andthen six drops of acetic acid were added. 10 mL of CH₂Cl₂ was added andthe mixture was kept in a freezer for 30 minutes. The mixture wasfiltered to remove solids and the filtrate was concentrated to a viscousresidue. Purification by silica gel chromatography (gradient 99/1 to96/4 CH₂Cl₂/MeOH) provided a solid which was triturated with MeOH togive 292 mg of a cream colored solid: ¹H (CDCl₃) 3.48 (m, 8H), 3.63 (bds, (CH₂CH₂O)_(n), integral too large to integrate), 3.98 (s, 4H), 4.18(bd m, 4H), 5.91 (bd m, 2H), 7.48 (bd m, 2H), 7.76 (d, 4H), 7.88 (d,4H), 9.38 (bd m, 2H): iodoacetyl determination (European Journal ofBiochemistry 1984, 140, 63-71): Calculated, 0.46 mmol/g; Found, 0.37mmol/g.

[0120] Conjugation of Peptides to Valency Platform Molecule #3 or #5:

[0121] All buffers were sparged with helium. The polyethylene glycol(PEG) derivative #3 or #5 was dissolved in a minimal volume(approximately 20 mg/mL) of 0.05 M NaHCO₃ (pH 8.25). Approximately 3equivalents of peptide were used per iodacetyl group on the PEGderivative. For para-aminobenzoic acid (PABA)-PEG, containing 2iodacetyl groups (MW=approximately 4100 g/mole), 6 equivalents ofpeptide were used for each equivalent of PABA-PEG. For diaminobenzoicacid (DABA)-PEG, containing 4 iodoacetyl groups (MW=approximately 4300g/mole), 12 equivalents of peptide were used for each equivalent ofDABA-PEG. The PEG solution was added to the reduced peptide solution andallowed to react for at least one hour in the dark. The peptideconjugate was purified by preparative HPLC. Before pooling andlyophilization, fractions were checked by electrophoresis using a 15%tricine gel. A description of the compositions of the five peptideconjugates is given in Table 3 and the structures are shown in FIG. 11.TABLE 3 Conjugates of melittin Peptides and PEG T cell # B cell Conju-activation Conjugate Valence Peptide epitopes/ gation by peptide ornumber platform conjugated molecule terminus conjugate¹ 1 5 6 2 Nno(pep) 2 3 6 4 N no(pep/conj) 3 3 7 4 C nd 4 3 5 4 N yes(pep) 5 3 8 8²C nd

EXAMPLE 6 Studies Using Melittin Peptide Conjugates to Tolerize MicePrimed and Boosted with Melittin

[0122] Murine Lymph Node Proliferation Assays

[0123] Food and water was provided ad libitum. Balb/c mice wereimmunized in each hind footpad with 50 μg of melittin molecule in CFA.Popliteal lymph nodes were harvested aseptically seven days later. Lymphnodes were gently dissociated by teasing the cells through a 50 meshsieve screen. The single cell suspension was washed in RPMI-1640 (IrvineScientific, Irvine Calif.) containing glutamine, penicillin andstreptomycin. 5×10⁵ cells in RPMI medium supplemented with 10% fetalbovine serum (FCS) in quadruplicate wells of round bottom 96-wellCorning microtitration plates were cultured with melittin or a melittinpeptide at 10, 1.0 or 0.1 μg/mL. Cells in the positive control wellswere cultured with murine interleukin 2 (IL-2) at 100 or 50 U/mL, PHA(phytohemagglutinin) at 1 μg/mL. The negative control wells containedlymph node cells in RPM-1640 and 10% FCS. The cells were cultured for 4days in a 37° C. incubator with 5% CO₂. Each well was pulsed with 1 μCiof [³H]thymidine (ICN Biochemicals, Costa Mesa, Calif.) for anadditional 18 hours. Cells were harvested onto a glass fiber filter matusing a semiautomatic cell harvester (Scatron, Sterling, Va.).Incorporation of [³]thymidine was determined by liquid scintillation.The results were expressed as average counts per minute.

[0124] In vivo Protocols

[0125] Balb/c mice were primed intraperitoneally (i.p.) with 4 μg ofmelittin in CFA. One month later the potential tolerogen or formulationbuffer was administered i.p. Three days later all mice received an i.p.injection of 4 μg of melittin in Incomplete Freund's Adjuvant (ICF)(Sigma Chemical Co., St. Louis, Mo.). 100 to 200 μL of blood wascollected from the retro-orbital venous plexus 10 days later. Serumsamples were assayed for anti-peptide, or anti-melittin, IgG antibodies.

[0126] Assay for IgG Anti-Melittin or Anti-Melittin Antibodies

[0127] An individual mouse's serum sample was assessed serially for thepresence of anti-melittin antibodies by ELISA. Falcon Probind 96-wellmicrotitration plates were precoated with 10 μg/mL melittin or melittinpeptide in phosphate buffered saline (PBS), pH 7.2, overnight at 4°. Theplates were washed twice with a wash solution containing PBS, 0.02%Tween-20, and 1% gelatin (Norland Products Inc., New Brunswick, N.J.).Plates were blocked with 200 μL PBS containing 5% gelatin for 1 hour at37°. Serum samples were prepared in a diluent of PBS containing 5%gelatin. Samples were tested at dilutions of 1:100 to 1:1000. After 1hour of incubation at 37° C., the plates were washed four times.ExtraAvidin peroxidase (Sigma Chemical Co., St. Louis, Mo.) was diluted1:1000 in PBS containing 5% gelatin. The plates were incubated 30minutes at 37° C. and then washed five times. Wells were developed withOPD (ortho phenylene diamine dihydrochloride, Sigma Chemical Co., St.Louis, Mo.) according to the manufacturer's directions, in the dark for15-30 minutes, and the reaction was stopped with 3 M HCl. The opticaldensity (OD) was determined at 450 nm on a microplate reader (Bio-tekInstruments, Winooski, Vt.).

[0128] Antibody Forming Cell Assay

[0129] Cellulose microtitration plates (Millipore Co., Bedford, Mass.)were prepared as indicated above for the IgG antibody (ELISA) assay.However, at the point in the assay where the serum samples were added tothe wells, splenic cells (5×10⁵/well) were added instead of serum, andincubated overnight. The remainder of the ELISA assay was performed asindicated above.

[0130] T Cell Epitopes

[0131] T Cells from mice primed with melittin showed T cellproliferation in response to the whole melittin molecule and toC-terminal melittin peptides 3, 4, and 5 (FIG. 6). However, C-terminalpeptides 1 and 2 induced no significant T cell proliferation. Melittinpeptides 6 and 5 were conjugated to PEG to make Conjugates 2 and 4,respectively. Like melittin peptide 2, the PEG conjugate of melittinpeptide 6 (Conjugate 2) also did not induce significant T cellproliferation. Mice treated with Conjugate 2 (10 mg/kg, 200 μg/mouse),had significantly lower levels of anti-melittin peptide 2 antibodies(FIG. 7) and also lower levels of anti-melittin antibodies (FIG. 8) ascompared to the control Balb/c mice treated with formulation buffer.Spleen cells from mice treated with buffer control or Conjugate 2 wereassayed for the ability of antibody-forming cells to produceanti-melittin or anti-melittin peptide 2 antibodies as measured in asoluble ELISA assay. As shown in FIG. 9, the levels of anti-melittinpeptide 2 antibody forming cells in the Conjugate 2 treatment group weresignificantly lower than in the control group which was administeredformulation buffer. Mice treated with Conjugate 4, a conjugate ofpeptide 5 (which contains a T cell epitope), failed to reduce the titerof antibodies to peptide 5 in treated mice. Thus, the conjugatecontaining a T cell epitope was not a tolerogen (FIG. 10). In fact,rather than reduce the response, the levels of anti-peptide antibody mayhave increased slightly.

EXAMPLE 7 Additional Studies Using Melittin Peptide Conjugates toTolerize Mice Primed and Boosted with Melittin

[0132] Female C57BL/6 mice, ages 5 to 8 weeks were purchased from TheJackson Laboratory, Bar Harbor, Me. Animals were maintained and treatedaccordingly to National Institutes of Health guidelines.

[0133] Immunization Protocol

[0134] Mice were primed by an i.p. injection containing 5 μg of melittinprecipitated on alum and 2×10⁹ B. pertussis as an adjuvant. The micewere boosted with 5 μg of melittin, i.p., in PBS.

[0135] pfc Assay

[0136] Sheep Red Blood Cells (SRBC) (Colorado Serum Co., Denver, Colo.)were conjugated with melittin-peptide 2 using carbodiimide. Fresh SRBC(less than 2 weeks old) were washed four times with cold saline and onetime with mannitol (0.35 M mannitol, 0.01 M NaCl). The SRBC weresuspended in mannitol to a concentration of 10% (v/v). 100 μL ofmannitol containing 30 μg of melittin peptide #3 were added to 1 mLaliquots of 10% SRBC which were then incubated on ice for 10 minutes.100 μL of a 100 mg/mL solution of1-ethyl-3(3-dimethylaminopropyl)-carbodiimide HCl (EDCI) was then addedand incubated on ice for 30 minutes. The SRBC were washed twice withBalanced Salt Solution (BSS) (Irvine Scientific Co, Irvine, Calif.) andresuspended to 10% (v/v). Lyophilized guinea pig complement (GIBCO, NewYork, N.Y.) was reconstituted with BSS and then diluted 1:3 with BSS.One mL of the diluted guinea pig complement was added to 3 mL ofconjugated SRBC. Rabbit anti-mouse IgG was added to give a finaldilution of 1:100 of the rabbit antiserum. This concentration waspredetermined to inhibit all IgM pfc while enhancing the maximum numberof IgG pfc. An equal volume of this complement/anti-mouse IgG/SRBCsuspension was mixed with a cell suspension of mouse spleen cells takenfrom a single mouse. 50 μL of each mixture was transferred to thechambers of a Cunningham slide (three chambers per slide). The edgeswere then sealed with paraffin and incubated at 37° C. for one hour. Thenumber of plaques per chamber was counted with the aid of a dissectingmicroscope. Each spleen suspension was also assayed using non-conjugatedSRBC as a control. The number of viable cells, in each spleen cellsuspension, was determined. The number of pfc per 10⁶ spleen cells wasdetermined for each chamber and the mean of the triplicates calculated.The number of pfc for non-conjugated SRBC was subtracted from the numberof pfc for conjugated-SRBC to determine the number of peptide-specificpfc.

[0137] Determining The Optimal Time to Measure pfc

[0138] Mice were primed with melittin. Groups (3 mice per group) ofprimed mice were boosted with melittin on days 2, 4, 6, and 8. On day 10the mice were sacrificed and their spleens harvested. Cell suspensionswere prepared and assayed for the number of peptide specific pfcdetermined. The optimal number of pfc was obtained 6 days after boostingwith melittin.

[0139] The Orientation of the Peptide on The PEG Conjugate does notAffect the Conjugate's Ability to Induce Tolerance

[0140] Two different tolerogens were constructed to determine if theorientation of the peptide on the PEG conjugate affects its ability toinduce tolerance. Peptide #7 (equivalent to peptide #2 plus C-terminalpenultimate insertion of Lys-Cys) was covalently bound to valencyplatform molecule 3 through its C-terminal end to make melittinConjugate 3. Groups (3/group) of mice primed with melittin were treated,.i.p., with conjugates or with saline. Five days later all of the mice,including the non-treated control group, were boosted with 5 μp ofmelittin. Six days later the mice were sacrificed, their spleens wereharvested and the number of peptide specific pfc determined. Asillustrated in Table 4, both orientations were effective in reducing thenumber of peptide-specific pfc/10⁶ spleen cells in mice primed andboosted with the parent protein melittin. TABLE 4 Orientation of thepeptide on the PEG conjugate does not affect the conjugates' ability toinduce tolerance Peptide specific Melittin pfc per 10⁶ spleen Conjugate#μg/mouse cells (Mean and S.D.) % Reduction 3 1000 μg  386 (85) 86.8% ″ 500 μg  489 (one mouse) 83.3% ″  250 μg  957 (298) 67.3% 2 1000 μg  546(160) 81.3% ″  500 μg  866.6 (235) 70.4% ″  250 μg 1280 (one mouse)56.2% None None 2924 (164) —

[0141] The Number of Peptides per PEG Conjugate Affects the Conjugate'sAbility to Induce Tolerance

[0142] Three different conjugates, each with a different number ofpeptides per PEG conjugate, were constructed to determine if the ratioof peptides to PEG molecule was important. Conjugate 1 had only twopeptides per PEG conjugate. Another had four peptides per PEG conjugate(Conjugate 2). The third had four, branched peptides (8 B cell epitopes)per PEG conjugate (Conjugate 5). Groups (3/group) of mice primed withmelittin were treated, i.p., with the different conjugates or withsaline. Five days later all of the mice, including the non-treatedcontrol group, were boosted with 5 μg of melittin. Six days later, themice were sacrificed, their spleens were harvested and the number ofpeptide-specific pfc determined. As shown in Table 5, Conjugate 1,containing two peptides per PEG molecule, was ineffective in reducingthe number of peptide-specific pfc/10⁶ spleen cells in mice primed andboosted with the parent protein melittin. The results show that bothmelittin conjugates 2 and 5 were effective as tolerogens; however,conjugate 5 which contained 8 epitopes (4 branched peptides) waseffective at a lower dose than conjugate 2, which contained fourunbranched peptides per valency platform molecule. TABLE 5 The number ofpeptides per PEG conjugate affects the conjugates' ability to inducetolerance Peptide specific Treatment Dose indirect IgG Molecule μg/mouse(nMoles) pfc(SD) % Reduction No treatment 1159 (280) std Conjugate 11000 (217) 1290 (98)  −11% 250 (54) 1350 (206) −16% Conjugate 2 500 (80) 585 (125) 49.5% 250 (40) 1001 (176)   14% Conjugate 5 500 (53)  630(325) 45.6%   250 (26.5)  443 (105) 61.8%   125 (13.25) 583 (69) 49.7%

[0143] Modifications of the above-described modes for carrying out theinvention that are obvious to those of ordinary skill in the fields ofimmunology, chemistry, medicine and related arts are intended to bewithin the scope of the following claims.

1 11 1 8 PRT Artificial Sequence Synthetic construct 1 Arg Ser Lys SerLys Ser Lys Cys 1 5 2 8 PRT Artificial Sequence Synthetic construct 2Ile Lys Arg Lys Arg Gln Gln Gly 1 5 3 9 PRT Artificial SequenceSynthetic construct 3 Trp Ile Lys Arg Lys Arg Gln Gln Gly 1 5 4 10 PRTArtificial Sequence Synthetic construct 4 Ser Trp Ile Lys Arg Lys ArgGln Gln Gly 1 5 10 5 11 PRT Artificial Sequence Synthetic construct 5Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly 1 5 10 6 12 PRT ArtificialSequence Synthetic construct 6 Cys Ile Ser Trp Ile Lys Arg Lys Arg GlnGln Gly 1 5 10 7 14 PRT Artificial Sequence Synthetic construct 7 XaaCys Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly Xaa 1 5 10 8 12 PRTArtificial Sequence Synthetic construct 8 Xaa Cys Trp Ile Lys Arg LysArg Gln Gln Gly Xaa 1 5 10 9 13 PRT Artificial Sequence Syntheticconstruct 9 Xaa Trp Ile Lys Arg Lys Arg Gln Gln Lys Cys Gly Xaa 1 5 1010 21 PRT Artificial Sequence Synthetic construct 10 Xaa Trp Ile Lys ArgLys Arg Gln Gln Trp Ile Lys Arg Lys Arg Gln 1 5 10 15 Gln Lys Cys GlyXaa 20 11 28 PRT Melittin from bee venom VARIANT 1 Xaa = This positionis H2N 11 Xaa Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu ProAla 1 5 10 15 Leu Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Xaa 20 25

1. A composition for inducing specific B cell anergy to an immunogenimplicated in an antibody-mediated pathology comprising a conjugate of anonimmunogenic valency platform molecule and at least one analog of theimmunogen wherein (a) the analog binds specifically to B cells to whichthe immunogen binds specifically and (b) the conjugate lacks a T cellepitope.
 2. The composition of claim 1 wherein the immunogen is anexternal immunogen.
 3. The composition of claim 2 wherein the externalimmunogen is a biological drug, an allergen or a D immunogen associatedwith Rh hemolytic disease.
 4. The composition of claim 1 wherein theimmunogen is a self-immunogen.
 5. The composition of claim 4 wherein theself immunogen is that associated with thyroiditis, diabetes, stroke,male infertility, myasthenia gravis, or rheumatic fever.
 6. Thecomposition of claim 1 wherein the immunogen and analog are of the samechemical class.
 7. The composition of claim 6 wherein the immunogen andthe analog are polypeptides.
 8. The composition of claim 1 wherein theimmunogen and the analog are of different chemical classes.
 9. Thecomposition of claim 1 wherein the valency platform molecule is apolymer.
 10. The composition of claim 9 wherein the polymer is acopolymer of D-lysine and D-glutamic acid.
 11. The composition of claim9 wherein the polymer is polyethylene glycol.
 12. The composition ofclaim 9 wherein the polymer is triethylene glycol.
 13. The compositionof claim 1 wherein the valency platform molecule has three to eightattachment sites.
 14. A pharmaceutical composition for treating anantibody-mediated pathology comprising a therapeutically effectiveamount of the conjugate of claim 1 combined with a pharmaceuticallyacceptable carrier.
 15. A method of inducing specific B cell anergy toan immunogen in an individual comprising administering to the individualan effective amount of the composition of claim
 1. 16. A method oftreating an individual for an antibody-mediated pathology in whichundesired antibodies are produced in response to an immunogen comprisingadministering a therapeutically effective amount of the composition ofclaim 1 to the individual.
 17. A method for making a conjugate usefulfor inducing specific B cell anergy to an immunogen implicated in anantibody-mediated pathology, the conjugate comprising a nonimmunogenicbiologically stable valency platform molecule and an analog of theimmunogen wherein (i) the analog binds specifically to B cells to whichthe immunogen binds specifically and (ii) the conjugate lacks a T cellepitope, comprising the steps of: (a) covalently bonding the analog ofthe immunogen lacking T cell epitopes to a nonimmunogenic valencyplatform molecule to form a conjugate; and (b) separating the conjugatefrom the reaction mixture.
 18. The method of claim 17 wherein theimmunogen is an external immunogen.
 19. The method of claim 18 whereinthe external immunogen is a biological drug or an allergen.
 20. Themethod of claim 17 wherein the immunogen is a self-immunogen.
 21. Themethod of claim 20 wherein the self-immunogen is that associated withthyroiditis, diabetes, stroke, male infertility, myasthenia gravis,rheumatic fever, or Rh hemolytic disease.
 22. The method of claim 17wherein the immunogen and analog are of the same chemical class.
 23. Themethod of claim 22 wherein the immunogen and analog are polypeptides.24. The method of claim 17 wherein the immunogen and analog are ofdifferent chemical classes.
 25. The method of claim 17 wherein thepolymer is a copolymer of d-lysine and D-glutamic acid.
 26. The methodof claim 17 wherein the polymer is a polyethylene glycol.
 27. A methodfor making a composition useful for inducing specific B cell anergy toan immunogen implicated in an antibody-mediated pathology, thecomposition comprising a pharmaceutically acceptable vehicle and aconjugate of a nonimmunogenic biologically stable valency platformmolecule and an analog of the immunogen wherein (i) the analog bindsspecifically to B cells to which the immunogen binds specifically and(ii) the conjugate lacks a T cell epitope, comprising the steps of: (a)covalently bonding the analog of the immunogen to a nonimmunogenicpolymer to form a conjugate; (b) separating the conjugate from thereaction mixture; and (c) combining the conjugate with apharmaceutically acceptable vehicle.